1. Field of the Invention
The present invention relates to a method for preparing a target-specific non-antibody protein, and more particularly, to a method for preparing a target-specific non-antibody protein comprising the steps of: selecting non-antibody proteins having a structural complementarity with the target site of a target protein in a non-antibody protein library; calculating a binding energy of the selected non-antibody protein and the target protein; selecting a non-antibody protein having a favorable binding energy among the selected non-antibody proteins; selecting amino acid residues having high binding energy among the interfacial amino acid residues of the selected non-antibody protein and the target protein; and substituting the selected amino acid residues with the amino acid residues having low binding energy. In addition, the present invention relates to a target-specific non-antibody specifically binding with EGFR (Epidermal Growth Factor Receptor) domain 2, which is prepared by the method, and a cancer therapeutic composition comprising the same.
2. Description of the Related Art
Conventionally, antibodies have been used as new therapeutic protein drugs. Antibodies are proteins produced by white blood cells called B lymphocytes in the immune system as a result of stimulation by an antigen. When antibodies encounter antigens, they recognize and bind to the antigens via receptors on the cells. Characteristically, antibodies specifically recognize and strongly bind to a particular protein. Due to this characteristic of binding to disease-causing proteins such as cancer, antibodies have been used for the purpose of preventing malicious protein interactions.
However, therapeutic antibodies have a large molecular size, their mass production is difficult, and their production process requires long processing times and high costs. Alternatively, recent studies have been actively conducted on non-antibody protein scaffolds for patient-tailored and targeted therapies. The non-antibody protein is a novel class of drug for patient-tailored and targeted therapies, and has begun to attract attention recently as an alternative to overcome the limitations of current therapeutic antibodies that has gained interest in present drug markets. Like therapeutic antibodies, the non-antibody proteins are aimed at treating various diseases such as cancer and autoimmune diseases or at preventing progression of diseases by selectively recognizing particular target molecules and strongly binding thereto so as to inhibit activity of the target molecules.
However, the existing non-antibody proteins are prepared relying on single scaffolds regardless of structures of the target molecules, like the conventional antibodies. Based on the tertiary structure, proteins bind to each other by interlocking together like Lego pieces. Thus, it is ideal to use tailored protein scaffolds specific to the particular targets. However, there are no technologies to find non-antibody proteins having a structural complementarity, capable of specifically binding to the target. Problematically, it is difficult to find target-specific non-antibody proteins used for the treatment of diseases. Experimentally, it is impossible to identify which proteins specifically bind to the particular target, because of diversity in the structure and type of proteins.
With respect to the existing antibodies and non-antibody proteins based on single scaffolds, their target site-binding region is accidentally determined, and thus the binding region cannot be designed in advance. In addition, the target sites bound by the known non-antibody proteins and antibodies are structurally restricted. It is fundamentally impossible to approach the concave target site. Inconveniently, a complex process for candidate screening, such as epitope mapping, should be performed for a long period of time, in order to find proteins capable of binding to a desired region of the target molecule among the target molecule-binding proteins.
In order to overcome the limitations of existing antibodies and non-antibody proteins, there is an urgent need to develop a technology for selecting scaffolds optimized for a target so as to design non-antibody proteins that bind to the desired target site of the target molecule. The technology for designing and preparing non-antibody proteins having a strong binding ability and specificity to the desired site of the target protein is a platform technology applicable to a wide range of target proteins, and this technology has not been realized in new antibody drugs or aptamers. Therefore, it is also urgently needed for the development of progressive patient-tailored and targeted therapies.
The present inventors have made an effort to develop a method for preparing target-specific non-antibody proteins. As a result, they found that virtual screening of a desired target site of a given target protein is performed based on a structural library of proteins with the predetermined tertiary structure so as to select an optimal protein scaffold having the highest structural complementarity, and randomization of amino acids that interfere with the binding to the target molecule is performed by phage display and bio-panning so as to prepare a target-specific non-antibody protein capable of optimally binding with the particular site of the predetermined target protein. Furthermore, they have prepared a non-antibody protein targeting the EGFR (Epidermal Growth Factor Receptor) domain 2 as a target for anticancer treatment, and examined its binding ability with the target, thereby completing the present invention.